SUMMARY OF WORK Na+/H+ exchanger (NHE) is one of the primary mechanism regulating intracellular pH through the exchange of extracellular Na+ for intracellular H+. Acute acidification of cells leads to an intrinsic mechanism for NHE activation by H+ binding to an intracellular site described as the H+ modifier site. All members of the NHE family respond to a variety of stimuli that include growth factors and cytokines, hormones,tumor promoters, osmotic stress, and other physiologic and pathophysiologic factors. Usually these factors change NHE activity by shifting the H+ modifier site sensitivity to pH. Many of these stimuli phosphorylate NHE by activating protein kinases. NHE activity can also regulated by Ca2+/calmodulin, ATP, and associated regulatory proteins. The nature and molecular mechanisms for this activation have not been examined, nevertheless, there are likely involve protein conformation changes. We used in our studies isolated vesicles from renal brush border plasma membranes containing the epithelial isoform, NHE3, involved in transepithelial reabsorption of NaCl and NaHCO3. Previous work in our laboratory suggests that the ion transporting activation of NHE by the H+ modifier site involves a transition from an inactive monomic form to an active oligomic (possibly a tetramer) form. Consistent with these earlier findings, the inactive NHE undergoes a slow (t1/2 2 minutes at 22oC) conformational transition to activated NHE after acid activation and that this activated state can be preserved permanently by glutaraldehyde cross-linking of the proteins. NHE also undergoes a slow conformational change from the active state to the inactive state after H+ removal from the modifier site (t1/2 20 minutes at 22oC). These processes were not significantly effected by a number of inhibitors for different kinases and phosphatases. In Western blots, glutaraldehyde cross-linking caused shifts in the apparent NHE molecular weights to high multiples of the monomer (2, 3, and 4 subunits) regardless of whether the cross-linking was done under H+ modifier site activating or inactivating conditions. Cross-linking also preserved the NHE activity present at the time of the cross-linking. We conclude that NHE likely exist in the membrane as an oligomer, but that intracellular acidification leads to slow conformational changes that functionally couple and activate those subunits. These experiment allow for the first time a method to examine separately intracellular proton effects on the H+ modifier site and the proton transport site. Changes in the rates of these slow conformational changes may have important implications for the regulation of intracellular pH by a variety of factors under both normal and pathological conditions.